Miller, Helen, Zhou, Zhaokun, Wollman, Adam J M orcid.org/0000-0002-5501-8131 et al. (1 more author) (2015) Superresolution imaging of single DNA molecules using stochastic photoblinking of minor groove and intercalating dyes. Methods. pp. 81-88. ISSN 1046-2023
Abstract
As proof-of-principle for generating superresolution structural information from DNA we applied a method of localization microscopy utilizing photoblinking comparing intercalating dye YOYO-1 against minor groove binding dye SYTO-13, using a bespoke multicolor single-molecule fluorescence microscope. We used a full-length ∼49kbp λ DNA construct possessing oligo inserts at either terminus allowing conjugation of digoxigenin and biotin at opposite ends for tethering to a glass coverslip surface and paramagnetic microsphere respectively. We observed stochastic DNA-bound dye photoactivity consistent with dye photoblinking as opposed to binding/unbinding events, evidenced through both discrete simulations and continuum kinetics analysis. We analyzed dye photoblinking images of immobilized DNA molecules using superresolution reconstruction software from two existing packages, rainSTORM and QuickPALM, and compared the results against our own novel home-written software called ADEMS code. ADEMS code generated lateral localization precision values of 30-40nm and 60-70nm for YOYO-1 and SYTO-13 respectively at video-rate sampling, similar to rainSTORM, running more slowly than rainSTORM and QuickPALM algorithms but having a complementary capability over both in generating automated centroid distribution and cluster analyses. Our imaging system allows us to observe dynamic topological changes to single molecules of DNA in real-time, such as rapid molecular snapping events. This will facilitate visualization of fluorescently-labeled DNA molecules conjugated to a magnetic bead in future experiments involving newly developed magneto-optical tweezers combined with superresolution microscopy.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | (c) 2015 Published by Elsevier Inc. This is an author produced version of a paper accepted for publication in Methods. Uploaded in accordance with the publisher's self-archiving policy. |
Dates: |
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Institution: | The University of York |
Academic Units: | The University of York > Faculty of Sciences (York) > Physics (York) The University of York > Faculty of Sciences (York) > Biology (York) |
Depositing User: | Pure (York) |
Date Deposited: | 02 May 2019 08:10 |
Last Modified: | 26 Nov 2024 00:29 |
Published Version: | https://doi.org/10.1016/j.ymeth.2015.01.010 |
Status: | Published |
Refereed: | Yes |
Identification Number: | 10.1016/j.ymeth.2015.01.010 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:145615 |
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